Ilaria Porcellato

A Retrospective Investigation on Canine Papillomavirus 1 (CPV1) in Oral Oncogenesis Reveals Dogs Are Not a Suitable Animal Model for High-Risk HPV-Induced Oral Cancer

CPV1 (also called COPV) is a papillomavirus responsible for oral papillomatosis in young dogs. The involvement of this viral type in oral oncogenesis has been hypothesized in oral squamous cell carcinomas (SCCs), but has never been investigated in other neoplastic and hyperplastic oral lesions of dogs. Aim of this study was to investigate the presence of CPV1 in different neoplastic and hyperplastic lesions in order to assess its role in canine oral oncogenesis; according to the results obtained, a second aim of the study was to define if the dog can be considered a valid animal model for oral high risk HPV-induced tumors. Eighty-eight formalin-fixed, paraffin-embedded (FFPE) canine oral lesions including 78 oral tumors (papillomas, SCCs, melanomas, ameloblastomas, oral adenocarcinomas) and 10 hyperplastic lesions (gingival hyperplasia) were investigated with immunohistochemistry for the presence of papillomavirus L1 protein and with Real-Time PCR for CPV1 DNA. RT-PCR for RNA was performed on selected samples. All viral papillomas tested were positive for immunohistochemistry and Real-time PCR. In 3/33 (10%) SCCs, viral DNA was demonstrated but no viral RNA could be found. No positivity was observed both with immunohistochemistry and Real-Time PCR in the other hyperplastic and neoplastic lesions of the oral cavity of dogs. Even though the finding of CPV1 DNA in few SCCs in face of a negative immunohistochemistry could support the hypothesis of an abortive infection in the development of these lesions, the absence of viral RNA points out that CPV1 more likely represents an innocent bystander in SCC oncogenesis. The study demonstrates a strong association between CPV1 and oral viral papillomas whereas viral contribution to the pathogenesis of other oral lesions seems unlikely. Moreover, it suggests that a canine model of CPV1 infection for HPV-induced oncogenesis could be inappropriate.

The contribution of stem cells to epidermal and hair follicle tumours in the dog

BACKGROUND Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. HYPOTHESIS Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. ANIMALS AND METHODS In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. RESULTS In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. CONCLUSIONS AND CLINICAL IMPORTANCE Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation.

CD3 and CD20 Coexpression in a Case of Canine Cutaneous Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides)

A 14-year-old female spayed Dachshund was presented with generalized scaling, erythema, pruritus, poor quality of hair coat, and progressive weight loss. Cutaneous epitheliotropic T-cell lymphoma (CETCL) was suspected. Skin biopsies were suggestive of CETCL. However, immunohistochemistry revealed the presence of numerous CD20+ and CD3+ cells. Clonality assay demonstrated a clonal T-cell receptor gamma rearrangement and a polyclonal IgH gene rearrangement. Double-label immunofluorescence confirmed coexpression of CD3 and CD20 by neoplastic cells. By double immunohistochemistry, neoplastic cells were CD3+ and PAX5–. The results are compatible with a CD3+, CD20+ CETCL. Coexpression of CD20 and CD3 has been recognized in peripheral T-cell lymphomas. Although documented in human CETCL, it has not been reported in canine CETCL. The pathogenetic basis of CD20 expression in mycosis fungoides is explored.

Feline Injection-Site Sarcoma

Feline injection-site sarcoma (FISS) is an aggressive tumor believed to arise from the proliferation of fibroblasts and myofibroblasts in areas of chronic inflammation, particularly at sites of injection. Local recurrence is frequent after surgical excision. Gelatinases (MMP-2 and MMP-9) and their inhibitor (TIMP-2) are endopeptidases pivotal in extracellular matrix remodeling and therefore in tumor invasiveness. The aim of this study was to investigate the immunohistochemical expression of MMP-2, MMP-9, and TIMP-2 in FISS to assess their usefulness as prognostic factors. Size, soft tissue sarcoma (STS) grading system, depth of infiltration, surgical margins, and Ki-67 index were evaluated as additional prognostic markers. Twenty-four cases of primary FISS were classified according to clinical follow-up as nonrecurrent (NR, n = 14; 58.3%) and recurrent (R, n = 10; 41.7%). MMP-2, MMP-9, and TIMP-2 were variably expressed in the FISS examined, confirming their role in tumor invasiveness, yet they did not show significant differences between the R and NR groups. These results could be due to different tumor stages or to the multiple activities of these enzymes, not limited to ECM remodeling. The immunohistochemical expression of these enzymes considered alone does not seem to be useful as a prognostic marker. STS grading system, depth of infiltration, surgical margins, and Ki-67 index did not relate to recurrence. Instead, the size of the tumor, measured after formalin fixation, with an optimal cutoff of 3.75 cm (accuracy = 86%; P < .05), and the mitotic count, with an optimal cutoff of 20 mitoses/10 HPF (accuracy = 80%; P < .05), could be evaluated as useful prognostic markers.

Feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling.

BACKGROUND Feline eosinophilic dermatoses (FEDs) are common diseases of cats with an unknown pathogenesis. They are histologically characterized by an eosinophilic infiltration and often by the presence of flame figures (FFs) and/or areas of loss of tissue architecture, here termed necrotic foci (NF). It has been postulated that an alteration in the degradation of the extracellular matrix could be responsible for these histological features. Matrix metalloproteinases (MMPs) are a group of proteases that are fundamental in extracellular matrix remodelling. HYPOTHESIS/OBJECTIVES The aim of the study was to investigate retrospectively the expression of a subgroup of MMPs, in particular MMP-2 and MMP-9 gelatinases, in FEDs. The expression of one of their inhibitors, TIMP-2, was also investigated in order to establish the role of these molecules in the pathogenesis of FEDs. The ultrastructural characteristics of extracellular matrix in FFs and NF were subsequently assessed. METHODS Fifty-one formalin-fixed, paraffin-embedded specimens from cutaneous and mucosal biopsies diagnosed as FEDs were investigated immunohistochemically. Two selected samples were processed for electron microscopy. RESULTS This study revealed an increased expression of MMP-2 in NF and a decreased expression of this gelatinase in FFs. An imbalance between MMP-2 and TIMP-2 was evident using immunohistochemistry. No significative results were observed for MMP-9 expression. Electron microscopy confirmed the lack of normal collagen fibres in NF, whereas in FFs only occasional, amorphous material was observed among normal collagen fibres. CONCLUSIONS AND CLINICAL IMPORTANCE Our study suggests that an imbalance in the expression of matrix metalloproteinases could be responsible for different morphological findings in FEDs. Further studies are needed to assess the role of matrix metalloproteinases in the pathogenesis of FEDs.

Canine cutaneous melanocytic tumours: significance of β-catenin and survivin immunohistochemical expression

BACKGROUND Recent investigations have highlighted the controversial role of Wnt/β-catenin pathway activation in human cutaneous melanoma. Survivin has been proposed as a valid prognostic marker for invasive and metastatic melanomas and lymph node melanoma metastasis in human cutaneous melanoma and is a promising therapeutic target. HYPOTHESIS/OBJECTIVES Our aim was to investigate the immunohistochemical expression of survivin and β-catenin in canine cutaneous melanocytic tumours, in order to understand their prognostic significance. METHODS Twenty-one melanocytic tumours (10 melanocytomas and 11 melanomas) were investigated by immunohistochemistry using specific anti-survivin and anti-β-catenin antibodies. A semi-quantitative method was used to analyse the results; β-catenin immunolabelling in neoplastic cells was evaluated as cytoplasmic, membranous or nuclear. The number of survivin-positive cells was counted within ~1000 neoplastic cells. Results were related to histopathological features, evaluated in haematoxylin- and eosin-stained slides, and to the clinical data obtained through a telephone survey with referring veterinarians. RESULTS Despite a low level of expression in the majority of cases, β-catenin was found to be correlated strongly with malignant behaviour (P < 0.01). An overexpression of nuclear survivin was statistically related to histological features of malignancy, presence of metastasis and death related to melanoma spread (P < 0.01). CONCLUSIONS AND CLINICAL IMPORTANCE The low nuclear β-catenin expression, mainly found in metastatic cases, would indicate that β-catenin activation may have only limited importance in the development or progression of canine cutaneous melanoma. The correlation of nuclear survivin expression with malignancy would indicate that survivin is possibly a useful prognostic marker and therapeutic target in canine melanoma patients.

Transcriptome Analysis of Canine Cutaneous Melanoma and Melanocytoma Reveals a Modulation of Genes Regulating Extracellular Matrix Metabolism and Cell Cycle

Interactions between tumor cells and tumor microenvironment are considered critical in carcinogenesis, tumor invasion and metastasis. To examine transcriptome changes and to explore the relationship with tumor microenvironment in canine cutaneous melanocytoma and melanoma, we extracted RNA from formalin-fixed, paraffin-embedded (FFPE) specimens and analyzed them by means of RNA-seq for transcriptional analysis. Melanocytoma and melanoma samples were compared to detect differential gene expressions and significant enriched pathways were explored to reveal functional relations between differentially expressed genes. The study demonstrated a differential expression of 60 genes in melanomas compared to melanocytomas. The differentially expressed genes cluster in the extracellular matrix-receptor interaction, protein digestion and absorption, focal adhesion and PI3K-Akt (phosphoinositide 3-kinase/protein kinase B) signaling pathways. Genes encoding for several collagen proteins were more commonly differentially expressed. Results of the RNA-seq were validated by qRT-PCR and protein expression of some target molecules was investigated by means of immunohistochemistry. We hypothesize that the developing melanoma actively promotes collagen metabolism and extracellular matrix remodeling as well as enhancing cell proliferation and survival contributing to disease progression and metastasis. In this study, we also detected unidentified genes in human melanoma expression studies and uncover new candidate drug targets for further testing in canine melanoma.

Oral Squamomelanocytic Tumour in a Dog: a Unique Biphasic Cancer

In human medicine, squamomelanocytic tumour is a malignant cutaneous neoplasm composed of closely intermingled neoplastic squamous cells and melanocytes. A multinodular gingival tumour in a 16-year-old, mixed breed neutered female dog was examined microscopically. Two populations of neoplastic cells, melanocytic and squamous epithelial cells were intermingled. The melanocytic cells were melan-A positive and cytokeratin AE1-AE3 negative and the squamous component was cytokeratin AE1-AE3 positive and melan-A negative. Bovine papillomavirus was not identified by immunohistochemistry or polymerase chain reaction. A diagnosis of squamomelanocytic tumour was made.

Feline herpesvirus ulcerative dermatitis: an atypical case?

BACKGROUND Feline herpesvirus ulcerative dermatitis is an uncommon skin disease in cats, with a predominantly facial distribution characterized by massive infiltration of eosinophils and, occasionally, predominant neutrophils. OBJECTIVE To describe the clinical and histopathological features of a putative atypical case of feline herpesvirus dermatitis. ANIMAL A 10-month-old, intact male, European cat was presented with chronic monolateral ulcerative dermatitis with adherent crusts on the left pinna. The lesion had been present for six months and worsened after the administration of corticosteroids. METHODS Clinical and histopathological examination, immunohistochemistry, nested PCR and transmission electron microscopy (TEM). RESULTS Histological examination of skin biopsies showed multifocal ulcerative and necrotic lesions, involving the superficial and deep dermis covered by thick haemorrhagic and serocellular crusts. The superficial, medium and deep dermis was heavily infiltrated with mast cells and plasma cells, with a lower number of neutrophils and eosinophils. In the nuclei of some cells in the deep dermis, whose histotype was unrecognizable with routine haematoxylin and eosin stain, intranuclear eosinophilic inclusion bodies were noticed. Nested PCR and TEM supported the hypothesis of FeHV-1-induced dermatitis. CONCLUSIONS AND CLINICAL IMPORTANCE This case is noteworthy for the infrequent location on the pinna and the atypical histopathological features of the lesion, with a predominant infiltration of mast cells and plasma cells. Our findings suggest that herpesvirus dermatitis should be listed as a differential diagnosis in case of ulcerative dermatitis when the location and histological features are atypical.

Reversible and cachexia‐associated feline skin fragility syndrome in three cats

Background Feline skin fragility syndrome (FSFS) is an acquired disorder characterized by altered collagen production resulting in an extremely thin and fragile skin. FSFS is associated with diseases characterized by excessive steroidal hormones that can inhibit collagen synthesis. It is also described concomitantly with severe inflammatory, infectious or neoplastic conditions where the pathogenesis remains largely unknown. Objectives To describe three cases of FSFS in cats that become cachectic secondary to different causes without glucocorticoid involvement. To describe the histopathological features of connective tissue for both fragile skin and the skin after healing. Results All cats developed cachexia in less than two months (body condition score ranging from 1–1.5). Concomitant diseases were diagnosed in Case 1 (aspiration pneumonia due to mega‐oesophagus) and Case 2 (feline immunodeficiency virus (FIV)). In Case 3, malnutrition was suspected as a primary cause. The main histological feature of fragile skin was an atrophic dermis with pale eosinophilic, thin and irregular collagen fibres with numerous red cores observed with Massons stain. Elastic fibres were normal. Postrecovery histopathological findings at 11 (Case 1) and six months (Case 3) after diagnosis, indicated normalization of the collagen and of the whole skin as compared with controls. Conclusions and clinical importance To the best of the authors’ knowledge, this is the first report describing a reversible, nonsteroid‐induced FSFS, associated with rapidly developing cachexia in cats.